Chloroquine Prevents T Lymphocyte Suppression Induced by Anthrax Lethal Toxin

Bacillus anthracis the causative agent of anthrax, produces a lethal toxin (LTX) that is a complex of 3 associated proteins: protective antigen (PA), lethal factor (LF), and edema toxin [1]. Receptors for PA are expressed on a wide variety of cells, making the latter sensitive to anthrax toxins. PA interacts with cell surface receptors, undergoes proteolytic activation, and forms membrane-penetrating heptamers binding LF and edema toxin [1]. These PA-LF complexes are internalized and, after acidification in phagolysosomes, dissociate, releasing the LF [1]. It has been shown that LF proteolytically cleaves several mitogen-activated protein kinase kinases (MAPKKs) and thereby interferes with signaling via MAPK cascades [2], disrupting regulation of major cellular functions

Toxins produced by B. anthracis kill or inactivate immune cells and thereby allow infection to progress [1, 3] The effects that LTX has on innate immunity (mainly on macrophages) are well documented (reviewed in [4]), whereas the response that the adaptive immune system has on the infection has still not been completely characterized. It has been demonstrated that LTX affects dendritic cell function, inhibits production of interleukin (IL)–2 in Jurkat cells, and interrupts signaling in human T lymphocytes [4–6]. As well, direct LTX-induced inactivation of T lymphocytes has been observed in vivo [7]

Modern antibacterial therapy does not provide complete protection against anthrax because of the severe systemic illness resulting from the toxins produced by B. anthracis during infection [3]. This fact indicates the need to look for new therapeutic approaches, such as attempts to affect key steps in the development of infection—for instance, the lysosomal processing of LTX. Chloroquine (CQ) is an agent that interferes with this processing, and it has been shown to protect isolated macrophages against the deadly effects of LTX [8]. However, no information concerning CQ-mediated protection of lymphocytes has been found. In the present study, we investigated the ability of CQ to reduce the suppressive effects that LTX has on T lymphocytes

Materials and methodsPeripheral-blood samples were obtained from healthy adult volunteers, according to the protocol approved by the Helsinki Committee of the Technion Faculty of Medicine. Peripheral-blood mononuclear cells were isolated by density-gradient centrifugation in the Histopaque 1077 system (Sigma-Aldrich) and, via adherence to plastic Petri dishes, were depleted from monocytes, to extract peripheral-blood lymphocytes (PBLs). The PBL fractions were >85% pure, and cell viability, as determined by propidium iodide (Sigma) staining and flow cytometry, was >95%. PBLs (10⁶ cells) were then resuspended in 0.2 mL of fresh RPMI 1640 culture medium (Biological Industries, Kibbutz Beth Haemek, Israel) and were incubated in 5% CO₂ at 37°C for different time periods. The cells were activated by CD3 cross-linking with 1 μg/mL mouse anti–human CD3 monoclonal antibody (MAb) (clone UCHT1; Serotec) and 1:100 rabbit anti–mouse IgG (Serotec). Recombinant PA, LF, and mutant LF (mLF) were purchased from List Biological Laboratories. PA was added to the cells at a concentration of 0.5 μg/mL, whereas LF was added at concentrations of 10, 100, 1000, and 10,000 ng/mL. In some experiments, CQ (Sigma) was added to the cells, at concentrations of 10, 50, and 100 μmol/L

After incubation, the cells were stained with MAb against CD3 (clone UCHT1; Serotec) and with MAb against CD69 (clone CH/4; Serotec), were rinsed, and then were analyzed by flow cytometry (FACSCalibur; Becton Dickinson), as described elsewhere [9]. Both the percentage of double-positive cells within the lymphocyte gate and the mean fluorescent intensity (MFI) were estimated, and an expression index, calculated by multiplying the percentage of double-positive cells by the MFI, was expressed in terms of arbitrary units (AU). Intracellular tumor-necrosis factor (TNF)–α and IL-10 were detected by use of a Cytofix/Cytoperm Kit (BD Pharmingen), as described elsewhere [9]. The experiments were repeated 6 times, each time with cells isolated from a different donor. The autofluorescence control and relative isotypic controls (Serotec) were analyzed in parallel. Staining with propidium iodide was used to determine the viability of the cells

After these treatments, cell extracts were prepared by use of protein-extraction reagent (Pierce Biotechnology), and aliquots of cell extract (30 μg of protein) were resolved by 10% PAGE under reducing conditions. After electrophoresis and transfer, proteins were identified by 2-step chemiluminescent detection (Pierce Biotechnology). Rabbit antibodies against the total and phosphorylated forms of p44/42 MAPK (i.e., extracellular signal–regulated kinase [ERK]1/2) and inhibitory κB (IκB)–α protein (Cell Signaling Technology) were used as primary antibodies. Films were analyzed by use of the Vilber Lourmat imaging system, and phosphorylation rates were calculated in terms of the fractions of total ERK and IκB-α proteins. The experiments were repeated 4 times, each time with cells isolated from a different donor

All data are presented as mean ± SEM. Comparison of the differences between groups (i.e., comparison of the independent variables) was performed by 1-way analysis of variance, followed by the Newman-Keuls post-hoc test. P<.05 was considered to be statistically significant

ResultsIn previous studies, CQ has been shown to protect both isolated macrophages [8, 10] and cultured epithelial cells [11] against the deadly effects of LTX. To determine whether blockade of lysosome acidification, which is required for release of LF from the PA-LF complex, might protect T lymphocytes too, we performed experiments in the absence and presence of CQ. Cross-linking of CD3 significantly increased CD69 expression on T lymphocytes—to 15.1±1.3 AU, versus 4.8±1.9 AU in untreated cells (P=.002). LTX significantly reduced CD69 expression, to 2.7±0.8 AU (P<.0001), indicating severe impairment of T lymphocyte activation. The specificity of this effect was demonstrated by employment of mLF, which was unable to cleave its cellular targets and therefore did not decrease CD69 expression (10.5±0.9 AU) (P<.0001, compared to the effect produced by native LF). No effect was found when either PA or LF was added separately to the cells (data not shown). CQ per se was found to have no substantial effect on T lymphocyte activation, whereas, in T lymphocytes exposed to LTX, increased concentrations of CQ restored the impaired expression of CD69 (figure 1A)

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Figure 1

Chloroquine (CQ)–induced protection against anthrax lethal toxin (LTX)–induced suppression of T lymphocytes. Flow-cytometric analysis of expression of CD69 (A) and tumor-necrosis factor (TNF)–α (B) by CD3⁺ cells activated in the absence (Activated) and presence of either 0.5 μg/mL protective antigen (PA) and 0.5 μg/mL lethal factor (LF) (in this case, LTX) or CQ at either 10 μmol/L (CQ10), 50 μmol/L (CQ50), or 100 μmol/L (CQ100); the expression index is expressed as the percentage of negative control. Data represent results of 4 separate experiments. Western blots (C) and densitometry assays (D) of phosphorylation of extracellular signal–regulated kinase (ERK)1/2 and IκB-α in peripheral-blood lymphocytes activated by cross-linking of CD3, in the absence (Activated) and presence of either 0.5 μg/mL PA and 0.5 μg/mL of native or mutant LF (mLF) (in this case, LTX or mLTX, respectively) or 100 μmol/L CQ (CQ100). Phosphorylation rates were calculated as the fraction of total ERK or IκB-α. Data from a representative experiment (n=4) are shown. Untreated cells served as the negative control. Results of analysis of variance, followed by Newman-Keuls test are as follows: *P<.05, vs. untreated; †P<.01, vs. activated; ‡P<.03, vs. LTX. p-ERK1/2, phosphorylated ERK1/2; t-ERK1/2, total ERK1/2

Just as it impaired CD69 expression, LTX also down-regulated the expression of TNF-α and IL-10, which mediate the counteracting pro- and anti-inflammatory responses, respectively (table 1). Addition of CQ to the cells significantly amplified expression of TNF-α (figure 1B). To better understand the mechanism of these favorable effects, we analyzed the intracellular signaling events. The LTX-induced suppression of T lymphocyte function appeared to be associated with abnormalities in intracellular signaling pathways such as MAPK and nuclear factor (NF)–κB. Although there was no change in overall expression of ERK and IκB-α proteins, LTX completely inhibited phosphorylation of ERK and IκB-α during 30 min of the exposure (figure 1C and 1D). The employment of mLF either negated or significantly reduced impairment of phosphorylation, suggesting that such impairment is LF specific (figure 1C and 1D). The addition of CQ to the culture medium negated the inhibitory effects of LTX and significantly improved the phosphorylation of ERK and IκB-α (figure 1C and 1D)

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Table 1

Suppression of cytokine expression in T lymphocytes by anthrax lethal toxin (LTX)

DiscussionIn the present study, LTX has been observed to interfere with signaling via MAPK and NF-κB cascades and to significantly impair T lymphocyte function. The main finding was that CQ has the ability to improve intracellular signaling and to prevent LTX-induced suppression of T lymphocytes

CQ is a widely used antimalarial agent that is suitable for treatment of chronic inflammatory and autoimmune diseases [12], and it also has been shown to interfere with the presentation and recognition of antigens by blocking the lysosomal processing of major-histocompatibility-complex molecules [12]. As well, intracellular Ca²⁺ flux and signaling via MAPK can be reduced by CQ (reviewed in [13]). Because lysosome acidification, which leads to dissociation of the internalized PA-LF complexes, is a key event in the pathogenesis of anthrax [11], agents interfering with this process may have practical applications. In fact, CQ has been shown to protect both macrophages [8] and epithelial cells [11] against LTX and has even been shown to increase the survival rate of LTX-treated mice [13]. Moreover, CQ-induced protection might be significantly augmented by inhibition of the furin-mediated activation of PA on the cell surface [10]; it has recently been demonstrated that this is a way in which macrophages can be protected by very low concentrations of CQ that are close to the plasma levels (2–5 μmol/L) seen after a therapeutic dose (10 mg/kg) of CQ has been administered [10]

The present study has observed that LTX inhibits T lymphocyte activation, as manifested by expression of CD69 and cytokines. CD69 is a C-type lectin that is expressed early and transiently by lymphocytes during their activation, whereas cytokines are versatile tools widely used by T lymphocytes for modulation of the immune response. The present study’s observation of reduced cytokine expression mediating both proinflammatory (in the case of TNF-α) and anti-inflammatory (in the case of IL-10) responses is consistent with the results of other studies, which have reported the inhibition of cytokine production in lymphocytes exposed to LTX [5–7]. These findings indicate severe impairment of T lymphocytes’ regulatory capacity in cases of anthrax. This view is also supported by a recent (2005) article in which Tournier et al. report simultaneous inhibition of secretion of both IL-10 and TNF-α in murine dendritic cells exposed to LTX [14]. That report’s results indicate that impaired cytokine production plays a critical role in the disruption of the host’s immunity

The present study observed, as expected, that LTX-induced suppression of T lymphocyte function is associated with MAPK inactivation, and this finding is fully consistent with data reported by other investigators, who have shown that LTX has a similar suppressive effect on MAPK in macrophages [2, 15], dendritic cells [4, 14], Jurkat cells, and normal lymphocytes [5–7]. In contrast to those of MAPK, the mechanisms of LTX-induced inhibition of NF-κB signaling are less well defined. Park et al. (2004) have demonstrated that LTX decreases, in a dose-dependent manner, IκB-α degradation in nonstimulated macrophages but that stimulation with lipopolysaccharide cancels this effect [15]. Because degradation of IκB-α reflects activation of the NF-κB pathway, these findings indicate that LTX’s effects on NF-κB signaling in macrophages are determined by additional stimuli. In Jurkat cells, LTX has been found to have no inhibitory effect on NF-κB signaling, in both resting- and activated-cell models [5]; in the present study, however, LTX inhibited phosphorylation of IκB-α in PBLs, indicating that the NF-κB pathway is involved in this dysfunction. These contradictory findings can be explained by the fact that these various studies used different models and methods, and it has already been demonstrated that Jurkat cells and normal lymphocytes respond differently to anthrax toxins [5, 6]

In lymphocytes exposed to LTX, CQ improved phosphorylation of ERK and almost completely restored phosphorylation of IκB-α. The significantly improved transduction of the appropriate signals normalized the expression of CD69 and cytokines. Most probably, these beneficial effects of CQ were related to its lysosomotropic activity. We have suggested that impaired lysosome acidification might prevent dissociation of PA-LF complexes and thereby might negate or greatly reduce the exposure of MAPKKs to LF. This suggestion is supported by the absence of any dramatic effects on CD3-mediated T lymphocyte activation that are due to CQ per se (figure 1). In the present study, the CQ-induced moderate reduction of phosphorylation of ERK, a finding that also has been reported by other investigations (reviewed in [13]), was compensated by the significantly increased phosphorylation of IκB-α, a fact that reflects activation of NF-κB and that correlates with intact expression of TNF-α (figure 1B). Currently we have no data elucidating the timing of these phenomena; this issue as well as the mechanism by which CQ counteracts LTX to inhibit phosphorylation of IκB-α is presently under investigation

In conclusion, the findings of the present study are the first to demonstrate that CQ improves intracellular signaling and protects T lymphocyte function against anthrax LTX. This effect may indicate a promising strategy in the treatment of anthrax

• Received March 14, 2006.
• Accepted May 19, 2006.

• © 2006 by the Infectious Diseases Society of America